The major etiological agent of posttransfusional and community acquired non-A non-B hepatitis has been identified as hepatitis C virus (HCV). Choo et al., Science 244:359-362 (1989). At present, intravenous drug abuse is the most important risk factor for transmission of HCV. However, different epidemiological studies have revealed that for up to 20 to 40% of patients chronically infected with HCV, no known risk factors for HCV can be demonstrated. Alter et al., N. Engl. J. Med. 327:1899-1905 (1992).
Although the disease associated with HCV may be benign, persistent infection may lead to liver cirrhosis and hepatocellular carcinoma (Saito et al., Proc. Natl. Acad. Sci. USA 87:6547-6549), although the mechanism of cellular transformation is unknown. HCV disease can be manifested as acute viral hepatitis which is usually clinically mild, but in other cases the disease may develop into a severe or fulminant hepatitis. Chronic HCV hepatitis is believed to occur more frequently than with hepatitis B virus, especially following posttransfusional acute hepatitis C disease, i.e., in about 54% of cases. Hollinger, in Fields Virology, 2d ed., Chpt. 78, eds. B. Fields and D. Knipe, Raven Press, NY (1990).
On the basis of sequence homology, the single-stranded positive-sense RNA enveloped HCV virus has been provisionally classified as a separate genus of the family Flaviviridae. Miller and Purcell, Proc. Natl. Acad. Sci. USA 87:2057-2061 (1990). The HCV genome is about 10 kb in length and it encodes a single polyprotein of about 3,000 amino acids that includes structural and nonstructural proteins that are processed by cellular and virus-encoded proteinases. The processed gene products include a putative capsid (C), three putative envelopes (E1, E2 type A, and E2 type B), and six nonstructural (NS) proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B). Representative sequences of HCV strains are described in U.S. Pat. No. 5,350,671 to Houghton et al., incorporated herein by reference.
Comparative sequence analysis of complete HCV genomes (Okamoto et al., Virology 188:331-341 (1992)) and PCR fragments from various genomic regions has shown that HCV may be grouped into distinct but related genotypes. At present, six major genotypes (1-6) with numerous subtypes (e.g., 1a, 1b; 2a, 2b, 3a, 5a) have been identified. Three additional types have been recently identified but are apparently limited in geographic distribution. Some genotypes have been associated with severity of disease (Pozzato et al., J. Med. Virol. 43:291-296 (1994)) and responsiveness to interferon therapy (Yoshioka et al., Hepatology 16:293-299 (1992)).
To date, treatment of HCV infection has primarily been with alpha-interferon. In some instances liver transplantation has been performed for end-stage hepatic deficiency, but invariably the transplanted liver also becomes infected with HCV and ultimately fails.
Gene therapy involves the introduction of genetic material into the cells of an organism to treat or prevent a disease. The material transferred can be from a few nucleotides to a few genes in size. Gene therapy is potentially useful in the treatment and prevention of acquired diseases, such as infectious diseases and cancer. A variety of cell types have been targeted in somatic cell gene therapy systems, including hematopoietic cells, skin fibroblasts and keratinocytes, hepatocytes, endothelial cells, skeletal and smooth muscle cells, and lymphocytes, each with varying success.
Methods for gene therapy involving hepatocytes have relied on gene transfer ex vivo, i.e., inserting genes into hepatocytes which have been removed from a patient which are then reimplanted into the liver, or in vivo, i.e., gene transfer directly into the liver. For ex vivo methods, gene transfer into cells must occur at high efficiency to obtain suitable numbers of cells for transplantation, because primary cultures of hepatocytes cannot be expanded. Long term expression in transduced hepatocytes has been accomplished with retroviral vectors, but the efficiency of transduction is relatively low (the retrovirus infects only dividing cells; Miller et al., Mol. Cell. Biol. 10:4239-4242 (1990)), and the protein may not be expressed in therapeutically or prophylactically effective amounts. In one ex vivo method approximately 20% of a patient's liver is surgically removed, the cells are then transduced with the retroviral vector, and then implanted back into the patient. This approach suffers from obvious disadvantages of surgical procedures and a low efficiency of transduction and expression of the gene product of interest.
Similarly, an in vivo approach to transducing hepatocytes with retroviral vectors involves first performing a partial hepatectomy followed by portal vein infusion of the vector. The removal of the majority of the liver is needed to stimulate liver regeneration so that the retrovirus will integrate into the cells' genomes. As with the ex vivo approach, this method suffers from requiring a major surgical procedure and under the best of conditions only about 1% of the liver mass contains the genetically modified vectors.
As an alternative to retroviral-mediated hepatic gene therapy, the adenovirus presents a transfer vector that can infect nonreplicating cells at high efficiency. Adenoviral DNA remains extra-chromosomal and thus is slowly lost from transduced hepatocytes over a period of several months. Li et al., Human Gene Ther. 4:403-409 (1993); Kay et al., Proc. Natl. Acad. Sci. USA 91:2353-2357 (1994). Additionally, a substantial portion of the adenovirus is taken up by organs and tissues other than the liver, which may raise issues of safety. (Smith et al., 1993, and Kay et al., ibid.). And, as adenovirus stimulates the production of neutralizing antibodies in an infected host, patients who have been naturally infected with adenovirus may be resistant to gene therapy using this vector, or secondary transductions may be prevented by the presence of antibodies produced in response to a primary transduction (Smith ibid., Kay, ibid.).
There remains a significant need in the art for compositions useful in treating hepatitis C infection and methods for their delivery to HCV-infected cells of the liver. Desirably, the compositions and methods should effectively reduce or eradicate HCV from infected cells, or should significantly impair the ability of the virus to replicate, thereby preventing further dissemination of the disease. The compositions should be inherently specific for HCV and of negligible toxicity. Quite surprisingly, the present invention fulfills these and other related needs.